Ribosomes, the cellular powerhouses of translation, are assembled from rRNA, the catalytic moiety, and scores of ribosomal proteins. In eukaryotic cells, the pre-rRNA undergoes extensive chemical modifications and several cleavage events as it is being assembled with ribosomal proteins into the small and large subunits of the ribosome. In the yeast, S. cerevisiae, progress over the last 15 years has revealed that these RNA processing events are mediated by numerous small nucleolar ribonucleoproteins (snoRNPs) and as many as 500 trans-acting protein and RNA-protein complexes. While by now most of the factors involved in making ribosomes have been identified, little is known about how they are assembled together to make the functional units of the pre-ribosome, and how each of these units contributes to specific events in gene expression. Our long-term goal is to understand the pre-rRNA processing, RNA folding and ribosome assembly steps essential to ribosome biogenesis in eukaryotic cells. The objective of this application is to determine the architecture and organization of some of the macromolecular assemblies involved in ribosome biogenesis in order to gain much needed mechanistic insights. The strong preliminary data that we have generated for each of these strategies indicates a high likelihood of success. The rationale for the proposed research is to attain an understanding of how the protein components of s(no)RNPs, RNPs and associated protein subcomplexes are assembled and how they interact with each other to mediate steps in ribosome biogenesis. For this study, we propose the following Specific Aims: 1. To test the hypothesis that the archaeal box C/D sRNP assembles and functions as a di-sRNP; 2. To elucidate the architecture and organization of small RNPs and protein complexes involved in eukaryotic SSU ribosome biogenesis; 3. To test the hypothesis that the pre-rRNA processing machinery is assembled in an ordered, stepwise manner to elucidate how these large macromolecules function in ribosome biogenesis. The proposed work is innovative because we will use experimental strategies that have not yet been applied to the study of ribosome biogenesis, and because little is known about the architecture and assembly of these ubiquitous RNA-protein complexes. The results will be significant because ribosome biogenesis is a fundamental step in gene expression to which all cells, particularly growing ones, devote a significant amount of their metabolism. Dysregulation of ribosome biogenesis is linked to cancer in humans, and mutations in SSU processome proteins have been linked to neonatal cirrhosis, infertility and neurofibromatosis.